Project Summary Iron-sulfur clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of iron-sulfur biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged, termed the NIF, ISC and SUF systems, and in each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular- level understanding of iron-sulfur cluster biosynthesis using the NIF, ISC and SUF systems. Elucidating the mechanism of iron-sulfur cluster biosynthesis is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, X-ray crystallography, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic and natural circular dichroism, resonance Raman, and Mssbauer) that can probe the nature and detailed properties of iron or iron-sulfur centers during cluster biosynthesis or transfer to acceptor proteins. The objectives are to establish the structure of cluster-bound forms and the molecular mechanism of cluster assembly and transfer for each the four known types of iron-sulfur cluster scaffold proteins, identify the roles and specificity of each type of scaffold protein in the maturation of iron-sulfur proteins, and characterize the mechanisms used to regulate iron-sulfur cluster biosynthesis. PUBLIC HEALTH RELEVANCE: The importance of iron-sulfur clusters to human health stems largely from their crucial role in iron homeostasis and their involvement in a large number of enzymes and proteins, particularly those in the mitochondrial respiratory chain. A molecular-level understanding of iron-sulfur cluster biogenesis is crucial for understanding a variety of human diseases involving anemias, myopathies and ataxias that arise from defects in iron-sulfur cluster assembly proteins.